Study of the brains functional anatomy has see tremendous progress in recent years, in part because of the availability of large databases of fMRI data obtained from human subjects at rest. However, the dependence of spontaneous brain activity patterns on physiological and behavioral conditions, as recently revealed by researchers in our lab and other labs, complicates interpretation. For this reason, our lab has continued to investigate the sources contributing to and factors affecting spontaneous brain activity. This has proceeded along three separate avenues, involving the analysis of existing data, as well as the initiation of novel experiments. In a first project, led by Xiao Liu, fMRI data from the human connectome project database was analyzed to investigate nature of spontaneous, widespread activity fluctuations that are generally observed in fMRI experiments. Based on previous research in the lab, it was hypothesized that such large scale activity may be the result of fluctuations in arousal state, and therefore involve basal forebrain and thalamus, brain regions along the classical arousal pathways. Averaging selected time frames of high global brain activity, it was found that basal forebrain, and to a lesser extend midline thalamic nuclei, where involved in the cortical activity patterns. Surprisingly, their activity counter-fluctuated with cortical activity, suggesting an inhibitory relationship. One possibility is that high arousal states may be associated with decreased cortical excitability resulting from feedback inhibition. Concurrent EEG-fMRI experiments are planned to further investigate this. In a related project, led by Catie Chang, we flowed up on a previous finding in macaque that behavioral arousal changes, as measured from eyelid position, are associated with a prototypical fMRI activity pattern. We have started a concurrent EEG-fMRI experiment in humans to relate the occurrence of the pattern to arousal state, as measured from the EEG signal. If this relationship is confirmed, this would allow identifying and separating arousal state dependent effects on brain activity from other types of spontaneous brain activity, thereby improving the charting of brain functional connectivity. Several test scans have been performed and data acquisition on 10-15 normal volunteers is just starting. A third project, led by Dante Picchioni, aims at investigating spontaneous brain activity across the broad spectrum of arousal states occurring during natural sleep. EEG, fMRI, and behavioral measures of arousal state are being acquired with the goals of better understanding the nature of spontaneous brain activity, its dependence on arousal state, an potential differences within classical, EEG-defined sleep states. Overnight experiments are being performed that are preceded with an adaptation session on the prior night. So far, 4 successful experiments have been performed and it is anticipated that data acquisition will be completed in the spring of 2017. Towards the quantification of iron and myelin I human brain, we have developed a novel MRI technique that is highly sensitive to the presence of proteins and lipids that make up myelin. Combined with previously developed techniques to generate magnetic susceptibility contrast, it appears possible to quantify both tissue iron and myelin in clinically feasible scan times. Preliminary evaluation of the technique has been performed in humans in selected brain regions and we are currently developing a version that facilitates whole-brain examination.